Culture independent analysis of greyback canegrub-associated microflora and microbial community comparison using subtractive hybridisation

Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putativelysaprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens. Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubsassociated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community.

Massively parallel sequencing and analysis of expressed sequence tags in a successful invasive plant

Background Invasive species pose a significant threat to global economies, agriculture and biodiversity. Despite progress towards understanding the ecological factors associated with plant invasions, limited genomic resources have made it difficult to elucidate the evolutionary and genetic factors responsible for invasiveness. This study presents the first expressed sequence tag (EST) collection for Senecio madagascariensis, a globally invasive plant species. Methods We used pyrosequencing of one normalized and two subtractive libraries, derived from one native and one invasive population, to generate an EST collection. ESTs were assembled into contigs, annotated by BLAST comparison with the NCBI non-redundant protein database and assigned gene ontology (GO) terms from the Plant GO Slim ontologies. Key Results Assembly of the 221 746 sequence reads resulted in 12 442 contigs. Over 50 % (6183) of 12 442 contigs showed significant homology to proteins in the NCBI database, representing approx. 4800 independent transcripts. The molecular transducer GO term was significantly over-represented in the native (South African) subtractive library compared with the invasive (Australian) library. Based on NCBI BLAST hits and literature searches, 40 % of the molecular transducer genes identified in the South African subtractive library are likely to be involved in response to biotic stimuli, such as fungal, bacterial and viral pathogens. Conclusions This EST collection is the first representation of the S. madagascariensis transcriptome and provides an important resource for the discovery of candidate genes associated with plant invasiveness. The over-representation of molecular transducer genes associated with defence responses in the native subtractive library provides preliminary support for aspects of the enemy release and evolution of increased competitive ability hypotheses in this successful invasive. This study highlights the contribution of next-generation sequencing to better understanding the molecular mechanisms underlying ecological hypotheses that are important in successful plant invasions.

Flexible airport terminal design : towards a framework

Flexibility is a key driver of any successful design, specifically in highly unpredictable environment such as airport terminal. Ever growing aviation industry requires airport terminals to be planned and constructed in such a way that will allow flexibility for future design, alteration and redevelopment. The concept of flexibility in terminal design is a relatively new initiative, where existing rules or guidelines are not adequate to assist designers. A shift towards flexible design concept would allow terminal buildings to be designed to accommodate future changes and to make passengers’ journey as simple, timely and hassle free as possible. Currently available research indicates that a theoretical framework on flexible design approach for airport terminals would facilitate the future design process. The generic principles of flexibility are investigated in the current research to incorporate flexible design approaches within the process of an airport terminal design. A conceptual framework is proposed herein, which is expected to ascertain flexibility to current passenger terminal facilities within their corresponding locations as well as in future design and expansion.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Challenges in passenger terminal design : a conceptual model of passenger experience

In recent years, de-regulation in the airline industry and the introduction of low-cost carriers have conspired to produce significant changes in the airport landscape. From an airport operator’s perspective, one of the most notable has been the shift of capital revenue from traditional airline sources (through exclusive use, long term lease arrangements) to passengers (by way of fees collected from ticket sales). As a result of these developments, passengers have become recognized as major stakeholders who have the power to influence airport profitability. This link between passenger satisfaction and profitability has generated industry wide interest in the “passenger experience”. In this paper, we define the factors which influence passenger experience, namely (a) artifacts, (b) services and (c) the terminal building, and explore the challenges that exist in the current approaches to terminal design. On the basis of these insights, we propose a conceptual model of passenger experience, and motivate its use as a framework for further research into improving terminal design from a passenger oriented perspective.

Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits

Background Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women. Methods Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR. Results Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence's of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6 %, 12.8 %, 4.6 % and 8.3 % respectively. Conclusions Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.

Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles

HIV-1 Pr55 Gag virus-like particles (VLPs) are strong immunogens with potential as candidate HIV vaccines. VLP immunogenicity can be broadened by making chimaeric Gag molecules: however, VLPs incorporating polypeptides longer than 200 aa fused in frame with Gag have not yet been reported. We constructed a range of gag-derived genes encoding in-frame C-terminal fusions of myristoylation-competent native Pr55Gag and p6-truncated Gag (Pr50Gag) to test the effects of polypeptide length and sequence on VLP formation and morphology, in an insect cell expression system. Fused sequences included a modified reverse transcriptase-Tat-Nef fusion polypeptide (RTTN, 778 aa), and truncated versions of RTTN ranging from 113 aa to 450 aa. Baculovirus-expressed chimaeric proteins were examined by western blot and electron microscopy. All chimaeras formed VLPs which could be purified by sucrose gradient centrifugation. VLP diameter increased with protein MW, from ∼100 nm for Pr55Gag to ∼250 nm for GagRTTN. The presence or absence of the Gag p6 region did not obviously affect VLP formation or appearance. GagRT chimaeric particles were successfully used in mice to boost T-cell responses to Gag and RT that were elicited by a DNA vaccine encoding a GagRTTN polypeptide, indicating the potential of such chimaeras to be used as candidate HIV vaccines. © 2008 Elsevier B.V. All rights reserved.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

Process based synthesis to evaluate design flexibility in airport terminal layout

Flexible design concept is a relatively new trend in airport terminal design which is believed to facilitate the ever changing needs of a terminal. Current architectural design processes become more complex every day because of the introduction of new building technologies where the concept of flexible airport terminal would apparently make the design process even more complex. Previous studies have demonstrated that ever growing aviation industry requires airport terminals to be planned, designed and constructed in such a way that should allow flexibility in design process. In order to adopt the philosophy of ‘design for flexibility’ architects need to address a wide range of differing needs. An appropriate integration of the process models, prior to the airport terminal design process, is expected to uncover the relationships that exist between spatial layout and their corresponding functions. The current paper seeks to develop a way of sharing space adjacency related information obtained from the Business Process Models (BPM) to assist in defining flexible airport terminal layouts. Critical design parameters are briefly investigated at this stage of research whilst reviewing the available design alternatives and an evaluation framework is proposed in the current paper. Information obtained from various design layouts should assist in identifying and defining flexible design matrices allowing architects to interpret and to apply those throughout the lifecycle of the terminal building.

Long terminal repeats act as androgen-responsive enhancers for the PSA-Kallikrein locus

The androgen receptor (AR) signaling pathway is a common therapeutic target for prostate cancer, because it is critical for the survival of both hormone-responsive and castrate-resistant tumor cells. Most of the detailed understanding that we have of AR transcriptional activation has been gained by studying classical target genes. For more than two decades, Kallikrein 3 (KLK3) (prostate-specific antigen) has been used as a prototypical AR target gene, because it is highly androgen responsive in prostate cancer cells. Three regions upstream of the KLK3 gene, including the distal enhancer, are known to contain consensus androgen-responsive elements required for AR-mediated transcriptional activation. Here, we show that KLK3 is one of a specific cluster of androgen-regulated genes at the centromeric end of the kallikrein locus with enhancers that evolved from the long terminal repeat (LTR) (LTR40a) of an endogenous retrovirus. Ligand-dependent recruitment of the AR to individual LTR-derived enhancers results in concurrent up-regulation of endogenous KLK2, KLK3, and KLKP1 expression in LNCaP prostate cancer cells. At the molecular level, a kallikrein-specific duplication within the LTR is required for maximal androgen responsiveness. Therefore, KLK3 represents a subset of target genes regulated by repetitive elements but is not typical of the whole spectrum of androgen-responsive transcripts. These data provide a novel and more detailed understanding of AR transcriptional activation and emphasize the importance of repetitive elements as functional regulatory units

miRDeep*: an integrated application tool for miRNA identification from RNA sequencing data

miRDeep and its varieties are widely used to quantify known and novel micro RNA (miRNA) from small RNA sequencing (RNAseq). This article describes miRDeep*, our integrated miRNA identification tool, which is modeled off miRDeep, but the precision of detecting novel miRNAs is improved by introducing new strategies to identify precursor miRNAs. miRDeep* has a user-friendly graphic interface and accepts raw data in FastQ and Sequence Alignment Map (SAM) or the binary equivalent (BAM) format. Known and novel miRNA expression levels, as measured by the number of reads, are displayed in an interface, which shows each RNAseq read relative to the pre-miRNA hairpin. The secondary pre-miRNA structure and read locations for each predicted miRNA are shown and kept in a separate figure file. Moreover, the target genes of known and novel miRNAs are predicted using the TargetScan algorithm, and the targets are ranked according to the confidence score. miRDeep* is an integrated standalone application where sequence alignment, pre-miRNA secondary structure calculation and graphical display are purely Java coded. This application tool can be executed using a normal personal computer with 1.5 GB of memory. Further, we show that miRDeep* outperformed existing miRNA prediction tools using our LNCaP and other small RNAseq datasets. miRDeep* is freely available online at http://www.australianprostatecentre.org/research/software/mirdeep-star

Supplementary Information : Hogan, Holland, Holloway, Petit and Read : Read Classification for Next Generation Sequencing, ESANN 2013, April 2013

This item provides supplementary materials for the paper mentioned in the title, specifically a range of organisms used in the study. The full abstract for the main paper is as follows: Next Generation Sequencing (NGS) technologies have revolutionised molecular biology, allowing clinical sequencing to become a matter of routine. NGS data sets consist of short sequence reads obtained from the machine, given context and meaning through downstream assembly and annotation. For these techniques to operate successfully, the collected reads must be consistent with the assumed species or species group, and not corrupted in some way. The common bacterium Staphylococcus aureus may cause severe and life-threatening infections in humans,with some strains exhibiting antibiotic resistance. In this paper, we apply an SVM classifier to the important problem of distinguishing S. aureus sequencing projects from alternative pathogens, including closely related Staphylococci. Using a sequence k-mer representation, we achieve precision and recall above 95%, implicating features with important functional associations.

Patients with terminal cancer who use alternative therapies: their beliefs and practices

This paper examines the extent to which patients who have been diagnosed as having terminal cancer choose to use non-medical therapies. In particular it is concerned with the illness behaviour of patients who are receiving conventional cytotoxic drug and radiation treatments, who also decide to use a wide range of ‘alternative’ medications and therapies. The paper discusses the findings of a study of 152 patients with metastatic cancer that examined the extent to which they used alternative cancer therapies, as well as the beliefs and attitudes they have about their cancer, its treatment, and the practitioners providing that treatment. Four groups of users of alternative therapies, who differ according to their commitment to and the type of therapies they use, were identified. Results of logistic regression analyses indicate that those using alternative therapies were different in range of social attitudes. These differences were primarily their greater reported ‘will to live’ and desire for control over treatment decisions, and the differing beliefs they hold about their disease.